ES2328077T3 - PROCEDURE FOR MODULATION OF THE PROLIFERATION OF MEDULAR THYROID CARCINOMA CELLS. - Google Patents

Abstract

A compound of the formula: Cpa-cycle (D-Cys-4-Pal-D-Trp-Lys-Thr-Cys) -NaI-NH2 or a pharmaceutically acceptable acid addition salt thereof.

Description

Proliferation modulation procedure of medullary thyroid carcinoma cells.

Background of the invention

It has been shown that somatostatin (SS), a Tetradecapeptide discovered by Brazeau et al., has potent inhibitory effects on various secretory processes in such tissues as pituitary, pancreas and gastrointestinal tract. The SS acts also as a neuromodulator in the central nervous system. These biological effects of SS, all inhibitory in nature, are stimulated through a series of receptors coupled to the G protein, of which five subtypes have been characterized different (SSTR1-SSTR5) (Reubi JC, et al., Cancer Res 47: 551-558, Reisine T, et al., Endocrine Review 16: 427-442, Lamberts SW, et al., Endocr Rev 12: 450-482, 4 Patel YC, 1999 Front Neuroendocrinology 20: 157-198). These five subtypes have affinities  similar for endogenous SS ligands but they have a distribution that differs in various tissues. Somatostatin is binds the five different receptor subtypes (SSTR) with affinity relatively high and equal for each subtype.

There is evidence that SS regulates cell proliferation by stopping cell growth by subtypes SSTR1, 2, 4, and 5 (Buscail L, et al., 1995 Proc Natl Acad Sci USA 92: 1580-1584; Buscail L , et al., 1994 Proc Natl Acad Sci USA 91: 2315-2319; Florio T, et al., 1999 Mol Endocrinol 13: 24-37; Sharma K, et al., 1999 Mol Endocrinol 13: 82- 90), or inducing apoptosis by subtype SSTR3 (Sharma K, et al., 1996 Mol Endocrinol 10: 1688-1696). SS and various analogues have been shown to inhibit normal and neoplastic proliferation in vitro and in vivo (Lamberts SW, et al., Endocr Rev 12: 450-482) by specific SS receptors (SSTR) (Patel YC, 1999 Front Neuroendocrinology 20: 157-198) and possibly different receptor actions (Weckbecker G, et al., Pharmacol Ther 60: 245-264; Bell GI, Reisine T 1993 Trends Neurosci 16: 34-38; Patel YC, et al., Biochem Biophys Res Commun 198: 605-612; Law SF, et al., Cell Signal 7: 1-8). In addition, there is evidence that different subtypes of SSTR are expressed in normal and neoplastic human tissues (Virgolini I, et al., Eur J Clin Invest 27: 645-647), conferring different tissue affinities with respect to various SS analogues and clinical response variable for its therapeutic effects.

The union has been associated with the different types of somatostatin receptor subtypes with the treatment of ailments and / or diseases. For example, the growth hormone inhibition to type 2 receptor of somatostatin ("SSTR2") (Raynor, et al., Molecular Pharmacol 43: 838 (1993); Lloyd, et al., Am. J. Physiol. 268: G102 (1995)) while insulin inhibition has been attributed to type 5 somatostatin receptor ("SSTR5") (Coy, et al. 197: 366-371 (1993)). Activation has been associated of types 2 and 5 with the suppression of growth hormone and more specifically with the adenomas that secrete GH (acromegaly) and adenomas that secrete TSH. Type activation has been associated 2 but not that of type 5 with the treatment of adenomas that secrete prolactin. Other guesses associated with activation of somatostatin receptor subtypes include the inhibition of insulin and / or glucagon to treat diabetes mellitus, angiopathy, proliferative retinopathy, the dawn phenomenon and nephropathy; inhibition of gastric acid secretion and more specifically peptic ulcers, enterocutaneous fistula and pancreaticocutaneous syndrome, irritable bowel syndrome, rapid gastric evacuation, liquid diarrhea syndrome, diarrhea related to AIDS, chemotherapy-induced diarrhea, acute or chronic pancreatitis and gastrointestinal tumors that secrete hormones; cancer treatment such as hepatoma; angiogenesis inhibition, treatment of disorders inflammatory such as arthritis; retinopathy, chronic rejection of allograft; angioplasty; preventing bleeding from the glass graft and gastrointestinal. It is preferred to have an analog that is selective for the specific subtype or subtypes of the receptor of the somatostatin responsible (s) for the biological response desired, thereby reducing interaction with other subtypes of the recipient that could cause side effects undesirable

Somatostatin (SS) and its receptors (SSTR1 a SSTR5) are expressed in human parafollicular C cells and cells of medullary thyroid carcinoma (MTC). MTC is a tumor that originates from thyroid parafollicular C cells that produce calcitonin (CT), somatostatin, as well as other diverse peptides (Moreau JP, et al., Metabolism 45 (8 Suppl. 1): 24-26). Recently, Mato et al. they showed that SS and SSTR were expressed in human MTC (Mato E, et al., J Clin Endocrinol Metab 83: 2417-2420). It has been documented that the SS and its analogues induce a decrease in plasma CT levels and symptomatic improvement in patients with MTC However, until now it had not been clearly demonstrated antiproliferative activity of SS analogues on tumor cells (Mahler C, et al., Clin Endocrinol 33: 261-9; Lupoli G, et al., Cancer 78: 1114-8; Smid WM, and col., Neth J Med 40: 240-243). In this way, the development and evaluation of analogues of the SSTR subtype selective regarding the growth of MTC cells provides a useful tool for clinical application. Until now no data has been reported concerning the implication specific subtype SSTR in the growth regulation of MTC cells.

This specification refers to the discovery that the human MTC TT cell line, which shows characteristics of MTC cells (Zabel M, et al., 1992 Histochemistry 102: 323-327, 2 Gagel RF, et al., 1986 Endocrinology 118: 1643-1651, Liu JL, et al., 1995 Endocrinology 136: 2389-2396) and that stably expresses all SSTR subtypes, responds to the activation of SSTR2 and SSTR5 by selective agonists of the subtype with two different models in terms of incorporating [3 H] thy and number of cells. SSTR2 suppress significantly the incorporation of [3 H] thy in the TT cells, that is, for example, inhibit DNA synthesis and reduce cell proliferation. Selective agonists for SSTR5 significantly increase the incorporation of [3 H] thy in TT cells, that is, increase the DNA synthesis, but alone fail to influence the cell proliferation In addition, SSTR2 antagonists counteract the action of agonists with preference for SSTR2 in TT cells. Even more, increasing concentrations of a SSTR5 selective agonist avoids dose-dependent manner suppression of the incorporation of [3 H] thy in the TT cells and proliferation produced by an agonist with preference for SSTR2, and vice versa, showing an antagonism between said agonists.

Interactions have recently been demonstrated hetero and homodimeric between subtypes of receptor families opiates (Jordan BA, et al., 1999 Nature 399: 697-700.) And SS (Rocheville M, et al., 2000 J. Biol. Chem. 275: 7862-7869). Cell studies cultured pituitary adenoma have shown that the subtype  2 and 5 of SSTR act synergistically in suppressing the growth hormone and prolactin secretion (Shimon I, and col., 1997 J. Clinical Invest. 100: 2386-2392, Jaquet P, et al., 2000 J Clin Endocrinol Metab. 85: 781-792). The finding that SSTR5 activation reduces SSTR2-mediated antiproliferative activity differs from the results in other tissues (Patel YC, 1999 Front Neuroendocrinology 20: 157-198, Buscail L, et al., 1995 Proc Natl Acad Sci USA 92: 1580-1584, Buscail L, et al., 1994 Proc Natl Acad Sci USA 91: 2315-2319, Sharma K, et al., 1996 Mol Endocrinol 10: 1688-1696). This is the first demonstration that SSTR subtypes 2 and 5 may act antagonistically in the cell growth regulation.

Thus, agonists with preference for SSTR2 and SSTR5 exert differential effects on the proliferation of the medullary thyroid TT cell line in vitro , according to their specific selectivity of SSTR. The proliferation of the TT cell line can be reduced by selective SSTR2 agonists, but not by SSTR5 agonists, and an SSTR5 agonist can avoid SSTR2-mediated anti-proliferative effects. The key inhibitory role of SSTR2 on the proliferation of MTC cells demonstrates that analogs that improve the affinity and selectivity of SSTR2 versus SSTR5 would be useful as antiproliferative agents in the treatment of MTC.

Summary of the Invention

In one aspect, the present invention is directed to a compound of formula: Cpa-cycle (D-Cys-4-PaL-D-Trp-Lys-Thr-Cys) -NaI-NH2,  or one of its pharmaceutically acid addition salts acceptable.

In a further aspect, the present invention refers to a compound of formula Cpa-cycle (D-Cys-4-Pal-D-Trp-Lys-Thr-Cys) -NaI-NH2,  or one of its pharmaceutically acceptable acid addition salts for use as a somatostatin receptor antagonist.

In a further aspect, the present invention refers to the use of a compound of formula Cpa-cycle (D-Cys-4-Pal-D-Trp-Lys-Thr-Cys) -NaL-NH2,  or one of its pharmaceutically acceptable acid addition salts as a somatostatin receptor antagonist.

In one embodiment, said antagonist of the somatostatin receptor is a selective antagonist of SSTR2.

Brief description of the drawings Figure 1 Intracellular calcium mobilization mediated by SSTR2 in vitro

CHO-K1 cells were harvested, expressing human SSTR2 as described in the section on Materials and Procedures and then the SS analogs (10-7 -10-6 M) for the measurement of intracellular Ca 2+ mobilization, expressed in relationship form between intracellular calcium concentration measured after the addition of SS analogues and baseline observed. The excitation and emission wavelengths were 340 and 510 nm, respectively. The data is represented as average ± DEM.

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Figure 2 Intracellular calcium mobilization mediated by SSTR5 in vitro

CHO-K1 cells were harvested, expressing the human SSTR5 as described in the section on Materials and Procedures and then the SS analogs (10-7 -10-6 M) for the measurement of intracellular Ca 2+ mobilization, expressed in relationship between intracellular calcium concentration measured after the addition of the SS analogs (Compound 1, Compound 5 and Compound 6) and the observed baseline value. Wavelengths of excitation and emission were 340 and 510 nm, respectively. The  data is represented as mean ± DEM.

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The structures of the compounds that appear In Figure 2 they are as follows:

Compound 1: D-NaI-cycle [Cys-Tyr-D-Trp-Lys-Val-Cys] -Thr-NH2;

Compound 2: cycle [Tic-Tyr-D-Trp-Lys-Abu-Phe];

Compound 3: 4- (2-Hydroxyethyl) -1-piperazinylacetyl-D-Phe-cycle (Cys-Tyr-D-Trp-Lys-Abu-Cys) -Thr-NH2;

Compound 4: 4- (2-Hydroxiethyl) -1-piperazine-2-ethanesulfonyl-D-Phe-cycle (Cys-Tyr-D-Trp-Lys-Abu-Cys) -Thr-
{} \ NH2 {2};

Compound 5: D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2; Y

Compound 6: Cpa-cycle (D-Cys-4-Pal-D-Trp-Lys-Thr-Cys) -NaI-NH2

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Figure 3 In vitro inhibition of SS-stimulated intracellular calcium mobilization by the SSTR2 antagonist

CHO-K1 cells were harvested, expressing the SSTR2 as described in the Materials section and Procedures, and then compound 6 was added (10 - 9 - 10 - 6 M) and SS (10 nM) for measurement of the effect of stimulated intracellular calcium mobilization by Compound 6 on SS (10-8 M), and expressed as percentage versus SS only. The excitation wavelengths and emission were 340 and 510 nm, respectively. The data is represent on average ± DEM.

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Figure 4 Expression of mRNA of somatostatin receptors in cells TT

The extracted RNA (1 µg / reaction) was treated with deoxyribonuclease and underwent reverse transcription using Oligonucleotide (dT) as primer. Samples incubated without RT enzyme served as a control. The aliquots of the CDNA generated and negative controls to amplification by subsequent PCR of the SSTRs, using the primers indicated in Table 1. The PCR products were resolved on a gel of 2% agarose. The products of the Expected PCR of SSTR1-5 (M band, marker of the PCR; G, PCR product of GAPDH amplification).

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Figure 5 Effect of SS analogues on the incorporation of [3 H] thy in TT cells

The cells were incubated in 24 plates wells for 48 hours in a culture medium supplemented with SS analogs at various concentrations (10-9, 10-8, 10-7, and 10-6 M). Control wells were treated with vehicle dissolution and the incorporation of [3 H] thy as radioactivity in the precipitated material with TCA. Data from six were independently evaluated individual experiments in quadruplicate and expressed as mean ± DEM of percent inhibition of incorporation of [3 H] thy versus untreated control cells * P <0.05 and ** P <0.01 versus control.

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Figure 6 Effect of SS analogues on cell proliferation TT

Cells were incubated in 96 plates wells for 48 hours in a culture medium supplemented with SS analogs at various concentrations (10-9, 10-8, 10-7, and 10-6 M). Control wells were treated with vehicle dissolution TT cell proliferation was measured as absorbance at 490 nm of each well. The data of six independent individual experiments with eight replicas expressed as the average ± DEM of the percentage of inhibition of cell proliferation versus non-control cells treated * P <0.05 and ** P <0.01 versus control.

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Figure 7 Effect of selective SSTR2 antagonist on incorporation from [3 H] thy to TT cells and cell proliferation during treatment with the SSTR2 agonist

Top panel: Cells were incubated in 24-well plates for 48 hours in a culture medium supplemented with 100 nM of Compound 1 or Compound 2, with or without the Compound 6 (10-7 M), Control wells were treated with vehicle dissolution The incorporation of [3 H] thy as radioactivity in the precipitated material with TCA. Data from six individual experiments were evaluated independently with four replicas expressed as the average ± DEM of the percentage of inhibition of incorporation of [3 H] thy versus untreated control cells * P < 0.05 and ** P <0.01 versus control.

Lower panel: cells were incubated in 96-well plates for 48 hours in a culture medium supplemented with 10-7 M of Compound 1 or compound 2 with or without Compound 6 (10-7 M). Control cells were treated with vehicle dissolution TT cell proliferation was measured as absorbance at 490 nm of each well. The data of six individual experiments with 0 replicas expressed as the average ± DEM of the percentage of proliferation inhibition cell versus untreated control cells * P <0.05 and ** P <0.01 versus control,

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Figure 8 Effect of the selective agonist of SSTR5 on the incorporation of [3 H] thy to TT cells and cell proliferation during treatment with the selective agonist of SSTR2

Top panel: Cells were incubated in 24-well plates for 48 hours in a culture medium supplemented with Compound 2 (10-7 M) with or without increasing concentrations of Compound 5 (10-9, 10-8, 10-7, and 10-6 M), or with Compound 5 (10-7 M) with or no decreasing concentrations of Compound 2 (10-6, 10-7, 10-8, and 10-9 M). The control wells are They treated with vehicle solution. The incorporation of [3 H] thy as radioactivity in precipitated material with TCA Data from six individual experiments were evaluated independently with four replicas expressed as the average ± DEM of the percentage of inhibition of incorporation of [<3> H] thy versus untreated control cells * P < 0.05 and ** P <0.01 versus control.

Bottom panel: Cells were incubated in 96-well plates for 48 hours in a culture medium supplemented with Compound 2 (10-7 M) without or with increasing concentrations of Compound 5 10-9, 10-8, 10-7, and 10-6 M), or with Compound 5 (10-7 M) with or no decreasing concentrations of Compound 2 (10-6, 10-7, 10-8, and 10-9 M). Control cells are treated with vehicle solution and the proliferation of TT cells as absorbance at 490 nm of each well. They were evaluated data from six individual experiments with eight replicates expressed as the average ± DEM of the percentage of inhibition of cell proliferation versus untreated control cells * P <0.05 and ** P <0.01 versus control.

Detailed description of the invention

Unless defined otherwise, all the technical and scientific terms used in this document they have the same meaning that a person commonly understands normally skilled in the art to which this invention belongs

Various receptors have been isolated from somatostatin (SSTRs), for example, SSTR-1, SSTR-2, SSTR-3, SSTR-4, and SSTR-5. In this way, a somatostatin agonist can be one or more of an agonist of SSTR-1, agonist of SSTR-2, SSTR-3 agonist, agonist of SSTR-4 or an SSTR-5 agonist. The which is understood by a type 2 receptor agonist of the somatostatin (i.e., SSTR-2 agonist) is a compound that (1) has a high binding affinity (for example, Ki less than 100 nM or preferably less than 10 nM or less than 1 nM) for SSTR-2 (for example, as defined by the receptor binding assay described below)  and (2) decreases the proliferation rate of the cells of the medullary thyroid carcinoma (for example, as shown by the biological test described below). What is meant by a selective somatostatin type 2 receptor agonist is a somatostatin type 2 receptor agonist that has a higher binding affinity (i.e., lower Ki) for SSTR-2 than for SSTR-5. What I know understood by a somatostatin type 5 receptor agonist is a somatostatin agonist that (1) has a high affinity binding (for example, Ki of less than 100 nM or preferably less 10 nM or less than 1 nM) for SSTR-5 (for example, as defined by the receptor binding assay described then and (2) attenuates the decrease induced by the agonist of SSTR-2 on the proliferation rate of  medullary thyroid carcinoma cells (for example, as sample by the biological test described below). The which is understood by a selective agonist of the type 5 receptor of Somatostatin is a type 5 agonist of somatostatin that has a higher binding affinity (i.e., less Ki) for SSTR-5 than for SSTR-2.

In one embodiment, the agonist of SSTR-2 is also a selective agonist of SSTR-2 In another embodiment, the selective agonist of SSTR-2 has a Ki value for SSTR-5 which is at least 2 times (for example, at at least 5 times or at least 10 times) greater than what you have for the SSTR-2 receiver (for example, as defined by the receptor binding assay described to continuation).

The agonist examples of SSTR-2 that can be used to carry out the Present invention include, but are not limited to:

D-NaI-cycle [Cys-Tyr-D-Trp-Lys-Val-Cys] -Thr-NH_ {2}, (Compound 1),

cycle [Tic-Tyr-D-Trp-Lys-Abu-Phe], (Compound 2),

4- (2-Hydroxyethyl) -1-piperazinylacetyl-D-Phe-cycle (Cys-Tyr-D-Trp-Lys-Abu-Cys) -Thr-NH_ {2}, (Compound 3), and

4- (2-Hydroxyethyl) -1-piperazine-2-ethanesulfonyl-D-Phe-cycle (Cys-Tyr-D-Trp-Lys-Abu-Cys) -Thr-NH2, (Compound 4).

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An example of an SSTR-5 agonist which can be used to carry out the present invention includes, but it is not limited to:

D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2. (Compound 5).

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Additional examples of agonists from the somatostatin are those covered by the formulas or those specifically listed in the publications shown at continuation.

European Application No. P5 164 EU (Inventor: G. Keri);

Van Binst , G. et al. Peptide Research 5: 8 ( 1992 );

Horvath , A. et al. Summary, "Conformations of Somatostatin Analogs Having Antitumor Activity", 22nd European Peptide Symposium, September 13-19, 1992 , Interlaken, Switzerland;

PCT Application No. WO 91/09056 (1991);

European Application No. 0 363 589 A2 (1990);

U.S. Patent No. 4,904,642 (1990);

U.S. Patent No. 4,871,717 (1989);

U.S. Patent No. 4,853,371 (1989);

U.S. Patent No. 4,725,577 (1988);

U.S. Patent No. 4,684,620 (1987);

U.S. Patent No. 4,650,787 (1987);

U.S. Patent No. 4,603,120 (1986);

U.S. Patent No. 4,585,755 (1986);

European Application No. 0 203 031 A2 (1986);

U.S. Patent No. 4,522,813 (1985);

U.S. Patent No. 4,486,415 (1984);

U.S. Patent No. 4,485,101 (1984);

U.S. Patent No. 4,435,385 (1984);

U.S. Patent No. 4,395,403 (1983);

U.S. Patent No. 4,369,179 (1983);

U.S. Patent No. 4,360,516 (1982);

U.S. Patent No. 4,358,439 (1982);

U.S. Patent No. 4,328,214 (1982);

U.S. Patent No. 4,316,890 (1982);

U.S. Patent No. 4,310,518 (1982);

U.S. Patent No. 4,291,022 (1981);

U.S. Patent No. 4,238,481 (1980);

U.S. Patent No. 4,235,886 (1980);

U.S. Patent No. 4,224,199 (1980);

U.S. Patent No. 4,211,693 (1980);

U.S. Patent No. 4,190,648 (1980);

U.S. Patent No. 4,146,612 (1979);

U.S. Patent No. 4,133,782 (1979);

U.S. Patent No. 5,506,339 (nineteen ninety six);

U.S. Patent No. 4,261,885 (1981);

U.S. Patent No. 4,728,638 (1988);

U.S. Patent No. 4,282,143 (1981);

U.S. Patent No. 4,215,039 (1980);

U.S. Patent No. 4,209,426 (1980);

U.S. Patent No. 4,190,575 (1980);

European Patent No. 0 389 180 (1990);

European Application No. 0505 680 (1982);

European Application No. 0 083 305 (1982);

European Application No. 0 030 920 (1980);

PCT Application No. WO 88/05052 (1988);

PCT Application No. WO 90/12811 (1990);

PCT Application No. WO 97/01579 (1997);

PCT Application No. WO 91/18016 (1991);

British Application No. GB 2,095,261 (1981); Y

French Application No. FR 2,522,655 (1983).

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Point out that for all agonists of the somatostatin described herein, each remainder of amino acid represents the structure of -NH-C (R) H-CO-, in the that R is the secondary chain (for example, CH 3 for Ala). The lines between amino acid residues represent bonds peptides that bind amino acids. Also, when the rest of amino acid is optically active, it is intended to be the configuration of form L unless expressly designated the form D. For clarity, disulfide bonds are not shown (for example, the disulfide bridge) that exists between two free thiols of Cys remains. The abbreviations of common amino acids are according to the recommendations of the IUPAC-IUB.

Synthesis of somatostatin agonists

The procedures for synthesizing agonists of somatostatin are well documented and understood within the capacity of a person normally skilled in the technique.

The synthesis of short amino acid sequences It is well established in the peptide technique. For example, the synthesis of HD-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH_ {2}, described above can be achieved by following the protocol shown in Example I of European Patent Application 0 395 417 A1. The synthesis of somatostatin agonists with a N-substituted term can be achieved, for example, by following the protocol defined in WO 88/02756, the Request European Patent No. 0 329 295 and PCT Publication No. WO 94/04752.

Any of the compounds herein Invention may have at least one asymmetric center. Can be additional asymmetric centers present in the molecule depending of the nature of the various substituents of the molecule. Every one of said asymmetric centers will produce two optical isomers and It is intended that all said optical isomers, whether separate, pure or partially purified optical isomers, racemic mixtures or diastereomeric mixtures thereof, whether included within the scope of the present invention.

Compounds can generally be isolated from the present invention in the form of its acid addition salts pharmaceutically acceptable, such as salts derived from using inorganic and organic acids. Examples of such acids are hydrochloric, nitric, sulfuric, phosphoric, formic, acetic, trifluoroacetic, propionic, maleic, succinic, D-tartaric, L-tartaric, malonic, methanesulfonic and the like. In addition, some compounds that they contain an acidic function such as carboxyl can be isolated in form of its inorganic salt in which the counterion can be select from sodium, potassium, lithium, calcium, magnesium and similar, as well as organic bases.

Pharmaceutically salts can be formed. acceptable by taking approximately 1 equivalent of an agonist from SSTR-2, for example, compound 1, and putting this in contact with about 1 equivalent or more of the acid appropriate appropriate salt that is desired. The preparation and the isolation of the resulting salt are well known for the person normally skilled in the art.

The compounds of this invention can be administer via oral, parenteral administration routes (for example, intramuscular, intraperitoneal, intravenous or subcutaneous, or implant), nasal, vaginal, rectal, sublingual or topical and can be formulated with pharmaceutical vehicles acceptable to provide appropriate dosage forms for Each administration route. According to this, the present invention includes within its scope pharmaceutical compositions that comprise, as active ingredient, at least one agonist of SSTR-2 in association with a vehicle pharmaceutically acceptable.

Solid dosage forms for Oral administration include capsules, tablets, pills, powders  and granules. In said solid dosage forms, the compound asset is premixed with at least one inert vehicle Pharmaceutically acceptable such as sucrose, lactose, or starch. Said dosage forms may also comprise, as is usual in practice, other different additional substances such as inert diluents, for example, lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, dosage forms can also comprise buffering agents. Can be prepared additionally tablets and pills with coatings Enteric

Liquid dosage forms for oral administration include emulsions, solutions, suspensions,  Pharmaceutically acceptable syrups, containing the elixirs inert diluents commonly used in the art, such as Water. In addition to said inert diluents, the compositions may also include adjuvants, such as wetting agents, agents emulsifiers and suspensions, and sweetening, flavoring agents and perfumes.

The preparations according to this invention for the parenteral administration include solutions, suspensions, or sterile aqueous or nonaqueous emulsions. The examples of solvents or non-aqueous vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may contain also adjuvants such as preservatives, humectants, emulsifiers, and dispersants. They can be sterilized by, by example, filtration through a filter that retains bacteria, incorporating sterilizing agents in the compositions, radiating the compositions, or heating the compositions. Be they can also prepare in the form of sterile solid compositions that can be dissolved in sterile water, or some other means Sterile injection immediately before use.

Compositions for rectal administration or vaginal are preferably suppositories that may contain, in addition to the active substance, excipients such as butter cocoa or suppository wax.

Compositions for nasal administration or sublingual are also prepared with standard excipients well known in the art.

In general, a dosage can be varied effective of the active ingredient in the compositions of this invention; however, it is necessary that the amount of the ingredient active be such that a suitable dosage form is obtained. The selected dosage depends on the desired therapeutic effect, of the route of administration, and of the duration of treatment, all of which is within the sphere of knowledge of a person normally skilled in the art. In general, it administer dosage levels between 0.0001 to 100 mg / kg of daily body weight to humans and other animals, for example, mammals

A preferred dosage range is 0.01 a 10.0 mg / kg body weight daily, which can be administered as a single dose or divided into multiple doses.

Materials and procedures

RT-PCR analysis was used to demonstrate that the RNAs of the five SSTR subtypes are expressed in the human MTC cell line, TT. It can evaluate the capacity of SS analogues with different affinities and specificities regarding subtypes of SSTR 2 and 5 to influence the proliferative activity of TT cells taking into account the incorporation of [3 H] thy, considered an indirect measure of the synthetic activity of DNA, and the number of viable cells.

All preferred agonists of SSTR2 were capable of significantly suppressing the number of TT cells to concentrations ranging between 10-9 M and 10-6 M. Compound 3 and Compound 4 significantly reduced (p < 0.05) [3 H] thy incorporation at 10-9 M but not at 10-8 M and 10-7 M, when its maximum effect was evident inhibitor on the number of cells. Each SSTR2 compound tested showed a tendency to decrease effectiveness with increasing concentration, however, the response curves in the form of Bell are common for SS. The inhibition of the incorporation of [<3> H] thy and the number of TT cells per Compound 1 and Compound 2 to 10-7 M was not associated with any action cytotoxic, as demonstrated by staining of Trypan Blue. In addition, this effect was completely counteracted by the simultaneous treatment of TT cells with Compound 6, a selective antagonist of SSTR2. Taken together, these results indicate that SS analogues with selectivity preferred by SSTR2 inhibited proliferation of TT cells that they specifically interacted with SSTR2. Cultivation of the line TT cells.

The American TT cell line was obtained Type Culture Collection (ATCC, Manassas, VA, USA). The line of TT cells is made up of parafollicular cells that produce Aneuploid transformed CT that are characterized by the presence of a TGC to TGG mutation (Cys to Trp) in exon 11 of codon 634 in the RET protooncogene (Cooley LD, et al., 1995 Cancer Genet Cytogenet 80: 138-149), a feature that the inventors They confirmed on the cell line they worked with. In addition, the TT cells show an unbalanced expression of the p53 gene tumor suppressor (Velasco JA, et al., 1997 Int J Cancer 73: 449-455). Immunohistochemistry studies demonstrated that TT cells express CT and CT receptor (Frendo JL, et al., 1994 FEBS Lett. 342: 214-216), the carcino-embryonic antigen (CEA), SS, the neurotensin, the peptide that releases gastrin (GRP), Leu and Met-enkephalin, the peptide that releases the hormone parathyroid (PTHrp), chromogranin A, SP-I, the synaptophysin, neuron specific enolasa (NSE), the receptor of 1,25-dihydroxyvitamin D 3, thyroxine hydroxylase, α-tubulin, and the cytokeratin (Zabel M, et al., 1995 Histochemical J. 27: 859-868). TT cells secrete an amount significant of CT and respond to changes in levels of ionized calcium (Zabel M, et al., 1992 Histochemistry 102: 323-327). In this way, the TT cell line is suitable for studies on parafolicular function and responses to endocrine and pharmacological stimuli.

The cells were kept in Nutrient Blend of Ham F12 with Glutamine (EuroClone Ltd, Torquay, United Kingdom), supplemented with 10% fetal bovine serum (FBS, Life Technologies, Milan, Italy), 100 U / ml penicillin, 0.1 mg / ml streptomycin, and 100 µg / ml amphotericin (EuroClone Ltd, Torquay, United Kingdom) at 37 ° C in a humidified atmosphere of 5% CO2 and 95% air. RNA isolation.

Total RNA was extracted from TT cells subconfluents using TRIZOL (Life Technologies, Milan, Italy). TRIZOL protocol is a modification of the extraction with guanidinium / phenol. Briefly, cultured cell media will aspirated and the cells washed with 1 X PBS. Reagent was added TRIZOL and the cells were lysed at room temperature for 10 min. Chloroform was added to the TRIZOL / cell lysate mixture, and it was let stand for 2-3 min, and then centrifuged at 12000 x g for 15 min. The aqueous layer of The centrifuged mixture. Isopropanol was added to precipitate the RNA, the residue was collected, washed with 75% ethanol and dried on air. Total RNA was resuspended in water treated with diethylpyrocarbonate (DEPC) and was quantified using UV spectrometry at 260 nm. To avoid DNA contamination, the RNA was treated with Ribonuclease-free deoxyribonuclease (Promega, Milan, Italy).

RT-PCR

Using a complementary DNA synthesis kit (First-line cDNA) (SuperScript Preamplification System for the synthesis of the First Chain cDNA, Life Technologies, Milan, Italy), 1 µg of total RNA was transcribed in a manner Reverse according to the manufacturer's protocol. The RT mix in tubes PCR was covered with 50 µl of white light mineral oil (Sigma-Aldrich Corp. Milan, Italy); It was carried out RT in the Minycicler (MJ Research Inc., Watertown, MA, USA) using a program with the following parameters: 10 min at 70 ° C, 1 min at 4 ° C, 5 min at 4 ° C. After supplementing with SuperScript II, it completed the reaction at 42 ° C for 50 min and then at 70 ° for 15 min. Samples were digested with RNase H (Promega, Milan, Italy) at 37 ° C for 20 min, and then stored at -20 ° C until the first PCR.

The cDNA was then amplified (1 µl of the reverse transcriptase reaction) by PCR with 1 U of Taq DNA polymerase (Life Technologies, Milan, Italy), in the conditions recommended by suppliers in a mixture of 50 µl reaction. After initial denaturation at 95 ° C for 5 min, the PCR reactions were carried out using oligonucleotide primers and related conditions in Table 1, describing the size of the expected fragments. Be analyzed the PCR products on a 2% agarose gel and visualized by staining ethidium bromide (ETB). For ensure that there was no contamination during the course of RT-PCR procedure, two types were prepared of negative controls. The first negative control was prepared omitting the total RNA in the RT. The second one was prepared replacing mixing cDNA with water in the PCR reaction. It was considered PCR was useful only if no bands were observed in the bands of negative control on a 2% agarose gel. Each product of the PCR was digested by restriction enzyme and was analyzed on a 2% agarose gel to further confirm the correct identification of the amplicons.

SSTR selective agonists and antagonists

The SS analogues are listed in Table 2 used in this study and their respective affinities with respect to different SSTR. Each compound, provided by Biomeasure Incorporated (Milford, MA, USA), was resuspended in acid 0.01 N acetic acid containing 0.1% bovine serum albumin (BSA) in order to provide uniform solubility and avoid non-specific binding to the various surfaces of the preparation. The specificity and selectivity of the analogues were determined by means of the Radioligand Bonding Test and they were transfected from stably way the CHO-K1 cells with each of SSTR subtypes, as follows.

The complete coding sequences of the genomic fragments of the genes of SSTR 1, 2, 3, and 4 and the clone of an SSTR5 cDNA were subcloned into the expression vector in pCMV mammals (Life Technologies, Milan, Italy). They were obtained clonal cell lines that stably expressed SSTRs 1-5 by transfection in cells CHO-K1 (ATCC, Manassas, Va, USA) using the simultaneous precipitation procedure with calcium phosphate (Davis L, et al., 1994 in: Basic Methods in Molecular Biology, 2nd Edition, Appleton & Lange, Norwalk, CT, USA: 611-646). Plasmid was included pRSV-neo (ATCC) as a selectable marker. Be selected clonal cell lines in RPMI 1640 media containing 0.5 mg / ml of G418 (Life Technologies, Milan, Italy), the ring was cloned, and expanded in the culture.

The membranes were obtained for in vitro receptor binding assays by homogenizing the CHO-K1 cells expressing the subtypes of SSTR in 50 mM Tris-HCl cooled in ice and centrifuging twice at 39000 g (10 min), with an intermediate resuspension In fresh buffer. The final residues were resuspended in 10 mM Tris-HCl for the assay. For tests of SSTRs 1, 3, 4, and 5, aliquots of the membrane preparations were incubated for 90 min at 25 ° C with 0.05 nM [125 I-Tyr11] SS-14 in 50 mM HEPES ( pH 7.4) containing 10 mg / ml BSA, 5 mM MgCl 2, 200 KIU / ml Trasylol, 0.02 mg / ml bacitracin, and 0.02 mg / ml phenylmethylsulfonyl fluoride. The final volume of the assay was 0.3 ml. For the SSTR 2 assay, [125 I] MK-678 0.05 nM was used as radioligand and the incubation time was 90 min at 25 ° C. incubations were terminated by rapid filtration through GF / C filters (pre-moistened in 0.3% polyethyleneimine) using a Brandel filtration sleeve. Each tube and filter were then washed three times with 5 ml aliquots of ice-cold buffer. Specific binding was defined as the total radioligand binding minus the binding in the presence of 1000 nM of SS-14 for SSTR 1, 3, 4, and 5, or 1000 nM of MK-678 for SSTR2.

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Biological Activity Evaluation

The biological activity of the SSTR selective agonists and antagonists by testing calcium mobilization in CHO-K 1 cells that they expressed the SSTR2 or the human SSTR5. The cells were harvested incubating in an EDTA / phosphate buffered saline solution 0.3% (25 ° C) and washed twice by centrifugation. The washed cells were resuspended in saline solution buffered with Hank (HBSS) to enter the Ca 2+ indicator Fura-2AM fluorescent. Cell suspensions (approximately 10 6 cells / ml) were incubated with 2 mM Fura-2AM for 30 min at 25 ° C. He withdrew fura-2AM not introduced by centrifugation two times in HBBS, and the final suspensions were transferred to a spectrofluorometer (Hitachi F-2000) equipped with a magnetic stirring mechanism and a bucket-type container With regulated temperature. After equilibration at 37 ° C, they were added SS analogues for the measurement of Ca 2+ mobilization intracellular The excitation and emission wavelengths were of 340 and 510 nm, respectively. In the cells that expressed SSTR2 (Figure 1), Compound 2 and Compound 1 stimulated significantly mobilization of intracellular Ca 2+ (indicated as the relationship between the stimulated value and the baseline) while Compound 6 did not, at concentrations rehearsed In addition, Compound 4 and Compound 3 were equally very powerful in stimulating the mobilization of Ca 2+. In cells expressing SSTR5 (Figure 2), the Compound 5 and Compound 1 significantly stimulated the mobilization of intracellular Ca 2+, while the Compound 6 showed a slight agonist activity in the range of 300 to 1000  nM. In cells expressing SSTR2 (Figure 3), Compound 6 inhibited SS-induced intracellular Ca 2+ mobilization in cells in cells expressing SSTR2 in a way dose-dependent with the complete suppression of the action of SS at approximately 10-7 M. Therefore, the evaluation of the mobilization of intracellular Ca 2+ showed that the activity biological of each of the various analogs was consistent with its receptor binding profile. DNA synthesis

The effects of the agonists were evaluated and selective SSTR antagonists on the DNA synthesis of TT cells determining the rate of incorporation of [<3> H] thymidine ([<3> H] thy), as described  previously (Davis L, et al., 1994 In: Basic Methods in Molecular Biology, 2nd edition, Appleton & Lange, Norwalk, CT, USA: 611-646, degli Uberti EC, et al., 1991 J Clin Endocrinol Metab 72: 1364-1371). The plates were plated TT cells in 24 multiwell plates (10 5 cells / well) and incubated for 48 hours in a medium supplemented with FBS at 10% in the presence of [3 H] thy (1.5 µCi / ml; 87 Ci / mmol) with or without each SS analogue at concentrations ranging from 10-6 to 10-9 M. The treatments were renewed by adding fresh analogues to the wells after the first 24 h of incubation, without removing the medium.

After incubation, three cells were washed times with ice-cold PBS and twice with acid 10% trichloroacetic (TCA) cooled on ice. The material TCA precipitate was solubilized in 500 µl of 0.2 mol sodium (l and 0.1% SDS. Then the radioactivity associated with cells in a spectrometer by scintillation. The results were obtained (counts per min per well) determining the average value of at least six quadruplicate experiments. The viability of the TT cells in control cultures and treated by staining of blue Trypan after 24 and 48 hours, and the number of viable cells It was almost 85-95%.

Cell proliferation

The effects of the agonists were evaluated and selective SSTR antagonists on cell proliferation TT through the Aqueous Non-Radioactive Cell Proliferation Assay CELL-TITER 96 (Promega, Milan, Italy), a colorimetric procedure to determine the number of cells viable in proliferation assays. The essay contains solutions of a tetrazolium compound (Owen reagent, MTS) and an electronic coupling reagent (phenazine methosulfate; PMS) MTS was bioreduced by formazan cells, which is soluble in tissue culture medium. You can measure the absorbance of the formazan at 490 nm directly on the plates of 96-well assay (Zatelli MC, et al., 2000 J Clin Endocrinol Metab 85: 847-852; Cory AH, et al., 1991 Cancer Commun 3: 207-212). The conversion of MTS into formazan Aqueous soluble is carried out by dehydrogenase enzymes which are found in metabolically active cells. The amount of formazan product as determined by the amount of absorbance at 490 nm is directly proportional to the number of live cells in the culture. Briefly, the cells were plated TT in 96 multiwell plates (2 x 10 4 cells / well) and they were incubated for 48 hours in a medium supplemented with FBS at 10% in the presence or absence of each SS analogue at concentrations ranging from 10-6 to 10-9 M. The treatments adding fresh analogs to the wells after first 24 h of incubation. At the end of the incubation period, added 20 µl of a combined solution of MTS / PMS to each well with an automatic pipette, and the plates were incubated for an additional 4 hours at 37 ° C in a 5% CO2 atmosphere humidified The absorbance at 490 nm was then recorded using an ELISA plate reader (EASIA Reader, Medgenix, Camarillo, CA). The results were obtained (absorbance at 490 nm) determining the average value of at least six experiments in Eight replicas

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Results SSTR expression in the human MTC TT cell line

To understand the individual role of SSTR2 and SSTR5 subtypes in the control of cell proliferation Parafollicular C, the inventors evaluated whether TT cells expressed the SSTRs that could mediate a potential response to the selective compounds for the individual SSTR subtypes. For answering this question, the inventors isolated the total RNA from cultured TT cells and carried out the reactions of the RT-PCR under the conditions described in Materials and Procedures The integrity of the cDNA was ensured by presence of the GAPDH signal. The absence of contamination was evaluated in genomic DNA in cDNA samples by the absence of any amplification in the PCR reaction using samples Transcribed in a non-reverse manner. It was found that amplification SSTR1, 2, 3, 4, and 5 positive in the examined cell line (Fig. 4) showed that these receptors were expressed in the line of TT cells of human MTC. The demonstration that the line of TT cells stably expresses the SSTR subtypes makes this system a suitable cellular model to evaluate the action of SS analogues selective for the recipient.

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Effect of selective SS analogues on incorporation of [3 H] thy in TT cells

The values of the incorporation of [3 H] thy with concentrations of 10-9 to 10-6 M of preferred agonists of SSTR2 (Compound 1, Compound 2, Compound 3, and Compound 4), the agonist preferred of SSTR5 (Compound 5) and the preferred antagonist of SSTR2 (Compound 6). As indicated, Compound 2 deleted significantly incorporating [3 H] thy in a 58-23% at concentrations ranging from 10-9 and 10-7 M. Compound 1 significantly suppressed the incorporation of [3 H] thy in a 41-21% at concentrations ranging from 10-9 and 10-6 M. The incorporation of [3 H] thy is also significantly reduced by Compound 4 (-13%, p <0.05) and Compound 3 (-17%, p <0.05) at 10-9 M. By on the contrary, Compound 5 significantly increased the incorporation of [3 H] thy into TT cells in a 80-175% The selective antagonist of SSTR2, the compound 6, does not alter the incorporation of [3 H] thy in TT cells compared to non control cells treated.

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Effect of selective SS analogs on proliferation TT cell

To examine in more detail the activity of SS analogues on the growth of TT cells, are It also analyzed its effect on the number of viable cells. Be represent in Figure 6 the effects of preferred agonists of SSTR2, a preferred agonist of SSTR5 and an antagonist preference of SSTR2 over the number of viable TT cells at concentrations ranging between 10-9 and 10-6 M. As indicates, all preferred compounds of SSTR2 inhibited significantly cell proliferation, when compared with untreated control cells at each concentration tested. The SSTR5 selective agonist, Compound 5, produced a slight increased proliferation of TT cells (up to 11% at 10-8 M), however, this does not represent a statistical difference regarding untreated control cells. The antagonist SSTR2 selective, Compound 6, does not seem to affect growth of the TT cells at the concentrations tested.

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The selective SSTR2 antagonist counteracts the effects of SSTR2 preferred agonist

To clarify further if SSTR2 is specifically involved in the mediation of the activity antiproliferative of the preferred agonists of SSTR2, it evaluated the incorporation of [3 H] thy and growth cell in TT cells exposed for 48 hours to Compound 1 and Compound 2, each alone (at 10-7 M) or in combination with Compound 6, a selective SSTR2 antagonist at concentration equimolar (10-7 M). The inhibition of the [3 H] thy incorporation induced by Compound 1 and Compound 2 by simultaneous treatment of TT cells with Compound 6 (Figure 7, top panel). The inhibition of TT cell proliferation induced by Compound 1 was reduced significantly from 46% to 10% by treatment concurrent with Compound 6. In addition, Compound 6 seemed completely block the antiproliferative activity of the Compound 2 (Figure 7, bottom panel). In this way, it was clearly demonstrated  the specific involvement of SSTR2 in mediating the effect inhibitor of an SSTR2 agonist on the proliferation of TT cells.

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Effect of the combination of a preferred agonist of SSTR2 with a preferred agonist of SSTR5 on the incorporation of [3 H] thy and cell proliferation

In order to analyze the effects of a SSTR2 and SSTR5 agonist in combination, the [3 H] thy incorporation and proliferation assaying each of Compound 2 and Compound 5 to 10-7 M in combination with increasing doses (from 10-9 M to 10-6 M) of the other compound. The results are summarized in Figure 8. He increased concentrations of the SSTR5 agonist (10-9 M at 10-6 M) avoided dose-dependent suppression of the incorporation of [3 H] thy to TT cells (Figure 8, upper panel) and proliferation (Figure 8, lower panel) produced by the SSTR2 agonist (10-7 M). These dates demonstrate an antagonism between effects mediated by SSTR5 and SSTR2 on proliferation.

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one

TABLE 2 Specificity of the receptor subtype of the human somatostatin (IC 50, nM)

2

Affinity was determined by subtype by binding assays of the membrane receptor to radioligand in Chinese hamster ovary cells expressing the cDNA of genes SSR2 or SSR5.

Claims (4)

1. A compound of the formula: Cpa-cycle (D-Cys-4-Pal-D-Trp-Lys-Thr-Cys) -NaI-NH2 or one of its pharmaceutically acid addition salts acceptable. 2. A compound of the formula Cpa-cycle (D-Cys-4-Pal-D-Trp-Lys-Thr-Cys) -NaI-NH2 or one of its pharmaceutically acceptable acid addition salts for use as a somatostatin receptor antagonist. 3. The use of a compound of the formula Cpa-cycle (D-Cys-4-Pal-D-Trp-Lys-Thr-Cys) -NaI-NH2 or one of its pharmaceutically acceptable acid addition salts as a somatostatin receptor antagonist. 4. The use according to claim 3, wherein said somatostatin receptor antagonist is an antagonist  selective for SSTR2.